7% formaldehyde in PBS, five minutes PBS, five minutes methanol, five minutes acetone and five minutes PBS or with 2% paraformaldehyde with 20 mM orthovanadate for twenty minutes followed by 2 x five mi nutes PBS washes. Coverslips this research were blocked with PBS 0. 02%Tween for five minutes and incubated with 1 150 MIB1 in PBS for one hour, or with 1 175 ER for 90 minutes. 1 100 ER 167. 1 400 pS2 for 90 minutes or 1 25 ER 118 overnight at room temperature. Unbound antibody was removed by washing with PBS. The EnVision system was used for visualization. Coverslips were washed in PBS and detected with diaminobenzidine tetrahydrochloride and hydrogen peroxide chromogen substrate and counterstained with methyl green. Immunostaining was evaluated at 20 magnification using an Olympus BH 2 light microscope and representative photographs were taken.
For Ki67, estimates of percentage of cells deemed positive versus negative equivocally stained were deter mined from five fields of view for each coverslip from three independent experiments. Sytox green viability assay A Sytox green viability assay was modified from Jones and Singer. Cells were seeded into 96 cell plates and left to adhere overnight. After 24 hours, Sytox Inhibitors,Modulators,Libraries green, a cell impermeant green fluorescent nucleic acid stain, was added to each control well and after one hour the number of cells with a compromised plasma membrane that had taken up the sytox green was counted using a fluorescent microscope. Cell membranes were permeabilised overnight with 0. 25% saponin in the presence of Inhibitors,Modulators,Libraries sytox green, and Inhibitors,Modulators,Libraries then total cell number well was counted.
The remaining experi mental cells were cultured for 72 hours in the presence of AZD8055, then the sytox Inhibitors,Modulators,Libraries green assay was performed to give a count for dead and total cell numbers as described above. Data were subsequently analysed by comparing live cell counts on day 0 with live cell counts on day 3. Cell death was considered to have occurred when the viable cell number fell below the day 0 pre treatment control number. Each treatment was assessed from eight replicates and three independent experiments. Migration assay Pore inserts were incubated with 300 ul sterile fibronectin in PBS for two hours at 37 C. Excess fibronectin was removed from the bot tom of the insert by washing in PBS. Inserts were air dried for 15 minutes, then 650 ul cell culture medium 25 nM AZD8055 was placed in the well and the insert suspended Inhibitors,Modulators,Libraries in it.
A total of 40,000 TamR cells in normal growth medium were added biological activity to the insert and the plate was incubated for 24 hours at 37 C. Cells on the insert were fixed with 3. 7% formaldehyde. Cells on the inside of the insert were removed with a cotton swab and cells that had mi grated to the lower side of the insert membrane were stained with 0. 5% crystal violet for 30 minutes. Excess stain was removed by repeated washing with distilled water.